Restoring of Glucose Metabolism of Engineered Yarrowia

Subscriber access provided by University of Florida | Smathers Libraries

Article

Restoring of Glucose Metabolism of an engineered Yarrowia lipolytica for Succinic Acid Production via a Simple and Efficient Adaptive Evolution Strategy Xiaofeng Yang, Huaimin Wang, Chong Li, and Carol Sze Ki Lin J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 05 May 2017 Downloaded from http://pubs.acs.org on May 7, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30

Journal of Agricultural and Food Chemistry

Restoring of Glucose Metabolism of an engineered Yarrowia lipolytica for Succinic Acid Production via a Simple and Efficient Adaptive Evolution Strategy

Xiaofeng YANG 1, 2, #, Huaimin WANG 2, #, Chong LI 2, Carol Sze Ki LIN 2, *

1

Guangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering,

School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, People’s Republic of China 2

School of Energy and Environment, City University of Hong Kong, Tat Chee Avenue,

Kowloon, Hong Kong

#

Authors contributed equally

*Corresponding author: Carol Sze Ki LIN, School of Energy and Environment, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, [email protected]

1 Environment ACS Paragon Plus


1

ABSTRACT

2

Succinate dehydrogenase inactivation in Yarrowia lipolytica has been demonstrated for

3

robust succinic acid production, whereas the inefficient glucose metabolism has hindered its

4

practical application. In this study, a simple and efficient adaptive evolution strategy via cell

5

immobilisation was conducted in shake flasks, with an aim to restore the glucose metabolism

6

of Y. lipolytica mutant PGC01003. After 21 days with 14 generations evolution, glucose

7

consumption rate increased to 0.30 g/L/h in YPD medium consisting of 150 g/L initial

8

glucose concentration, while poor yeast growth was observed in the same medium using the

9

initial strain without adaptive evolution. Succinic acid productivity of the evolved strain also

10

increased by 2.3 folds, with stable cell growth in YPD medium with high initial glucose

11

concentration. Batch fermentations resulted in final succinic acid concentrations of 65.7 g/L

12

and 87.9 g/L succinic acid using YPD medium and food waste hydrolysate, respectively. The

13

experimental results in this study show that a simple and efficient strategy could facilitate the

14

glucose uptake rate in succinic acid fermentation using glucose-rich substrates.

15 16

KEYWORDS: Adaptive evolution, Cell immobilisation, Glucose metabolism, Succinic acid,

17

Yarrowia lipolytica


Page 2 of 30

Page 3 of 30


18

1. INTRODUCTION

19

The increasing energy demand and cost of petroleum have spurred the need for a shift from

20

petroleum refinery to bio-based economy.1 This trend requires the development of highly

21

efficient utilization processes to exploit fully the potential of agricultural residues or food

22

supply chain waste into chemicals, materials and fuels.2 Succinic acid (SA) was identified as

23

one of the top twelve potential chemical building blocks by the US Department of Energy.3, 4

24

The SA market size was estimated at US$ 191 million in 2013, production volume is

25

expected to grow rapidly.5 However, the practical application of the natural producers in SA

26

fermentation have the following disadvantages: (i) sensitivity of microorganisms;

27

(ii) expensive nutrient requirements; (iii) complicated product recovery and purification; (iv)

28

large amount of wastes generated.6, 7 In the last decade, intensive research effort has been

29

made in metabolic engineering of Escherichia coli for SA production.6 However,

30

fermentation of E. coli faces several challenges,6,

31

bacteriophage infections, fermentation at near-natural pH, complicated downstream

32

processing and rigorous carbon catabolite repression.

8

including the susceptibility to

33 34

Recently, metabolic engineering of yeast for SA production has received significant scientific

35

interest as an alternative strategy. In contrast to prokaryotes, yeast is highly tolerant of low

36

pH values and generally recognised as safe (GRAS), making it superior for industrial

37

application.9,10

38

reconstruction12 and microarray gene transcription analysis13 were integrated with metabolic

39

engineering to improve SA production and cell growth rate of Saccharomyces cerevisiae.

40

Although considerable research progress has been made, none of the engineered yeast

41

S. cerevisiae have yet achieved commercial application.14 Yan et al.9 used another strategy by

42

using a potentially high yield pathway (i.e. via the reductive TCA pathway) with a theoretical

Metabolic

profiling

analysis,11

genome-scale


metabolic

network


43

maximum glucose to SA yield of 1.71 mol/mol, but only 0.3 mol/mol SA yield on glucose

44

was achieved. To date, the highest SA titer achieved by engineered S. cerevisiae fermentation

45

was 43 g/L, which was reported in the US patent by Van De Graaf et al.15

46 47

Yuzbashev et al.10 demonstrated a strictly aerobic yeast Yarrowia lipolytica that could be an

48

alternative SA producer via oxidative TCA pathway, in which a maximum SA titer of 17.4

49

g/L was obtained without pH control. Regulation of the SDH2 expression allowed the mutant

50

to produce 25 g/L SA under oxygen limitation conditions.16 Moreover, Kamzolova et al17-19

51

developed a two-stage process for succinic acid production, which involved the combined

52

microbial synthesis of α-ketoglutaric acid (KGA) by Y. lipolytica and the subsequent

53

decarboxylation of KGA by hydrogen peroxide to SA. Under strictly controlled conditions,

54

up to 71.7 g/L succinate was produced from ethanol with the yield of 70%, and 69.0 g/L

55

succinate was produced from rapeseed oil.17, 18 Our former studies have demonstrated the

56

possibility of SA fermentation from glycerol-based medium by deletion of Ylsdh5 encoding

57

succinate dehydrogenase subunit 5 in Y. lipolytica.20,21 By using in-situ fibrous bed bioreactor

58

(isFBB), up to 198.2 g/L SA was accumulated from crude glycerol using this strain.21

59

So far, all the metabolic engineering of Y. lipolytica for SA production has led to partial or

60

total loss of its ability to grow in glucose-based medium, which limits its industrial

61

application. This glucose metabolism problem of engineered strain for SA production has

62

been reported also in E. coli and S. cerevisiae.6 Methods such as increasing the reducing-

63

force,22 intracellular ATP controlling,23 NADH/NAD+ ratio regulation24 by metabolic

64

engineering or adaptive evolution

65

metabolism. Compared to other three methods, adaptive evolution is more suitable for the

66

built-in metabolic pathway.22, 25 Adaptive evolution in immobilised cell mode by FBB has

67

been demonstrated as a highly efficient system for improvement of glucose uptake, alcohol

22, 25

have been attempted in order to improve the glucose


Page 4 of 30

Page 5 of 30


68

and organic acid productivity and tolerance, as compared to the conventional free cell

69

fermentation.26-28 However, operation conditions include monitoring of FBB bioreactor by

70

highly-skilled labour, large volume of feeding medium and long-duration of fermentation

71

process are essential for operating immobilised cell mode by FBB, makes it far from

72

industrial application.

73 74

On the other hand, glucose-rich feedstocks are the most important carbon source in microbial

75

fermentation.29 As vast majority of the carbon fixed by plant photosynthesis is available in

76

the form of sugar polymers consist of cellulose, hemicellulose and starch.

77

utilisation of glucose-rich feedstocks such as agro-waste residues and food waste have been

78

investigated extensively to improve the economic competitiveness of bio-based SA

79

production.2, 30 An alternative strategy is the integration of waste-based biorefinery concept,

80

as food waste contains valuable nutrients and our former study successfully demonstrated the

81

production of glucose-rich hydrolysate using food waste as feedstock.2 Herein, a simple and

82

efficient adaptive evolution strategy was evaluated to enhance the glucose uptake rate of

83

Y. lipolytica PGC01003 in SA fermentation. The evolved strain was investigated on the

84

subsequent SA fermentation using glucose-rich food waste hydrolysate in bench-top

85

fermenter. In this study, the technical feasibility of SA production using food waste

86

hydrolysate and engineered Y. lipolytica was examined. The results would demonstrate the

87

potential using a low-cost feedstock for succinic acid fermentation as an environmentally

88

friendly and economically sound bioconversion process.

89 90

2. METHODS AND MATERIALS

91

Strains and media


Therefore,


92

The engineered strain of Y. lipolytica PGC01003, in which the Ylsdh5 genes encoding

93

succinate dehydrogenase was knocked out for SA production in the previous work.20 The

94

seed culture was stored in 30% (v/v) glycerol at -80 oC, which was activated in 50 mL

95

modified YPG medium containing (w/v): 1% yeast extract, 2% tryptone, and 2% glycerol at

96

28 oC and 220 rpm. The yeast strains were cultivated in YPG or YPD with various initial

97

concentrations of glycerol or glucose at 28 oC and 220 rpm. Carbon and nitrogen sources

98

were prepared separately and sterilized at 121 oC for 15 minutes.

99 100

Food waste handling and hydrolysis

101

Mixed food waste was collected from restaurants in Hong Kong Science Park. The storage

102

and handling method was described by Sun et al.31 and the composition of food waste was

103

determined as described in our previous study.32 Food waste with a solid-to-liquid ratio of

104

50% (w/v) was hydrolysed in a 2.5-L bioreactor (BioFlo/CelliGen 115, New Brunswick

105

Scientific, Edison, NJ, USA) at 55 oC. In order to control the nutrient composition of food

106

waste hydrolysate being used in experiments, a consortium of commercial enzymes kindly

107

provided by the Shangdong Longda Bio-Products Co., Ltd. were added at the beginning of

108

hydrolysis. The consortium of enzymes contained (per gram dry food waste): Glucoamylase

109

(120 U), amylase (10 U) and protease (150 U) .31 Samples were taken every 2-3 hours for

110

sugar and free amino nitrogen (FAN) analyses. Finally, the hydrolysate was sterilised by

111

membrane filtration (0.22 µm) and stored at -20 oC before use.

112 113

Shaking flasks fermentation of Y. lipolytica

114

To compare Y. lipolytica fermentation in YPD and YPG media, shaking flasks fermentation

115

was conducted in 250-mL shake flasks consisted of 50-mL YPD or YPG medium at 28 oC

116

and 220 rpm. The fermentation began with inoculation of 1 mL of seed culture, and 1 mL


Page 6 of 30

Page 7 of 30


117

broth was sampled every few hours for cell dry weight (CDW) measurement and high

118

performance liquid chromatography (HPLC) analysis.

119

To assess the stability of the evolved strain PSA02004, the cells were first transferred to YPG

120

medium with 20 g/L initial glycerol concentration, and then 1 mL of the culture was

121

transferred into YPD medium with 150 g/L initial glucose. The fermentation was carried out

122

in 250-mL shake flasks with 50-mL YPD or YPG medium at 28 oC and 220 rpm. The

123

fermentation began with addition of 1 mL of seed culture, and 1 mL broth was sampled every

124

few hours for CDW measurement and HPLC analysis.

125 126

Adaptive evolution of Y. lipolytica to glucose-rich YPD medium

127

A series of experiments using adaptive evolution in either free cell fermentation or

128

immobilised cell fermentation were conducted in 250-mL shake flasks with 50-mL YPD

129

media containing various initial glucose concentrations, and shaking incubation at 220 rpm

130

and 28 oC. To obtain higher cell biomass, 10 g/L of glycerol was supplemented into 1% YPD

131

in the zero generation. Then it was transferred into 2.5% YPD medium which was marked as

132

the first generation. Once the OD600 reached 1.8-2.2 within 24 hours, the culture was

133

transferred into YPD medium with higher glucose concentration, and the initial glucose

134

concentration increased stepwise from 25, 50, 75, 100 to 150 g/L.

135 136

In free cell fermentation, the inoculum ratio of Y. lipolytica PGC01003 was 5% (v/v). In

137

immobilised cell fermentation, 1% (w/v) of absorbent cotton was added in the culture to

138

adsorb and immobilise cells in the first generation. Then, the absorbent cotton with

139

immobilised cells was transferred to the fresh medium in the subsequent generations. After

140

14 generations of adaptive evolution, 0.2 mL of the cell culture was plated on solid YPD

141

medium containing 150 g/L glucose, and single colonies were selected. Six single colonies of



142

each fermentation mode were picked up and screened by monitoring the cell growth rate in

143

YPD medium with 150 g/L initial glucose concentration. The initial strain PGC01003 was

144

used as a control.

145 146

Batch fermentation

147

A 2-L Sartorius Biostat B benchtop fermenter (B. Braun Melsungen AG, Melsungen,

148

Germany) with 1.0 L working volume was used in batch fermentation. The pH value was

149

automatically controlled at 6.0 with the addition of 5 mol/L NaOH, and the foaming was

150

controlled with the addition of antifoam A (Sigma, Germany). Bench-top scale fermentations

151

were performed at 28 oC, stirring speed at 600 rpm and aeration rate at 2.0 L/min using either

152

defined medium or food waste hydrolysate.

153 154

Analytical methods

155

The analytical methods have been described in our previous work.20 Microbial growth was

156

determined by OD600 and CDW. The specific growth rate (µ) was calculated by Equation (1):

157

= ×

(1)

158 159

where X is DCW and t is fermentation time.

160 161

Glucose and metabolite concentrations were quantified using the ultra-performance liquid

162

chromatography (UPLC) system (Waters, MA, USA), which was equipped with an Aminex

163

87H column (Bio-Rad, Hercules, CA, USA) and a refractive index detector. H2SO4 (5 mM)

164

was served as mobile phase at a flow rate of 0.6 mL/min. Temperature of column and

165

detector were kept at 60 oC and 35 oC, respectively. All samples and mobile phase were


Page 8 of 30

Page 9 of 30


166

filtered by 0.22-µm membrane before loading. The FAN concentration of the hydrolysate was

167

analysed using the ninhydrin colorimetric method.33

168



169

3. RESULTS AND DISCCUSSION

170

Adaptive evolution by free cell and immobilised cell modes

171

As shown in Table 1, PGC01003 grew well in YPG medium and consumed 18.6 g/L glycerol

172

to produce 5.4 g/L SA with 4 g/L cell biomass. Whereas only 5.7 g/L glucose was consumed

173

in YPD medium after 84 hours, and the SA titer and CDW were only 35% and 31% of that in

174

YPG medium, respectively. Disruption of succinate dehydrogenase in Y. lipolytica not only

175

accumulated SA, but also caused partial loss of glucose metabolism ability.16, 20 The FAD

176

recycled into FADH2 in the oxidation of SA into fumaric acid by SDH in the native

177

Y. lipolytica, which has been disturbed in the SDH inactivated mutant (Fig. 1).34,35 Glycerol

178

metabolism could restore the FAD/FADH2 recycle via the phosphorylation and successive

179

oxidation reaction into dihydroxyacetone phosphate (DHAP).36 Moreover, the fast growth in

180

glycerol could be caused by more transporters for glycerol uptake than that for glucose

181

uptake in Y. lipolytica.36 Comprehensive metabolic and transcriptomic analyses also indicated

182

the glucose transporters are not enough in Y. lipolytica.37 Yuzbashev et al. (2016) suggested

183

acidification could negatively affect the cell growth of yeast on glucose-based media.

184 185

Therefore, this would limit the practical application of Y. lipolytica PGC01003 in SA

186

fermentation using naturally derived sugar-rich substrates. As most of the renewable

187

feedstocks, such as corn straw, wheat straw and food waste are glucose-rich in nature as they

188

predominantly consist of cellulose and starch as sugar polymers. Therefore, in order to

189

enhance the glucose uptake rate in microbial SA fermentation, adaptive evolution of Y.

190

lipolytica PGC01003 was performed. A highly efficient cell immobilisation method using

191

cotton as absorbent was developed in this study. After cell growth to exponential phase, they

192

were then transferred to the fresh medium. Free cell fermentation was carried out as a control

193

group. Moreover, a rational design in composition of cultivation medium was applied for the


Page 10 of 30

Page 11 of 30


194

initial medium, which was supplemented with 10 g/L glycerol. This design could facilitate

195

higher biomass production in the first generation, which is the key factor for a successful and

196

efficiently evolved adaptive process. After 14 transfers, the microbial growth in YPD

197

medium with 150 g/L glucose in both fermentation modes were observed (Fig. 2). The

198

immobilised cell fermentation took 21 days, which was 3 days shorter than that of free cell

199

fermentation. The final evolved strains were designated as PSA01 and PSA02 for the free cell

200

fermentation and immobilised cell fermentation, respectively.

201

In the free cell fermentation, the glucose uptake rate was only 0.07 g/L/h in the third

202

generation with medium containing 50 g/L initial glucose concentration, which increased four

203

times to 0.29 g/L/h by PSA01 with 150 g/L initial glucose concentration. With the similar

204

procedure, the glucose uptake rate increased three times to 0.30 g/L/h by PSA02. This high

205

sugar consumption rate would lead to significant improvement of cell growth and SA

206

productivity. Compared to the third generation, the cell growth rate and SA productivity

207

increased by 2.9 and 4.0 times using the PSA01 strain, which were 4.8 and 5.0 times higher

208

than those fermentation using the PSA02 strain. Moreover, the cell growth rate also

209

significantly increased 2.2 and 2.3 times in free cell fermentation and immobilised cell

210

fermentation, respectively. This suggests adaptive evolution by both fermentation modes

211

could enhance the glucose uptake rate, the cell growth rate and SA productivity. Additionally,

212

this immobilised cell fermentation has proved to be successful and highly efficient for

213

adaptive evolution of Y. lipolytica.

214 215

Evaluation of the evolved Y. lipolytica strains

216

The evolved strains of PSA01 and PSA02 were cultivated separately on solid YPD agar

217

plates with 150 g/L glucose for screening single colonies with stable phenotype. Six single

218

colonies from both PSA01 and PSA02 strains were selected randomly for investigation of



219

adaptive evolution in shake flask fermentation. As shown in Table 2, rapid cell growth was

220

observed in cultivation broth consisting of YPD medium with 150 g/L initial glucose

221

concentration, and 2.78-2.95 g/L CDW was obtained after 24 hours (Table 2). These results

222

also indicated that the adaptive evolution was feasible and efficient.

223 224

As shown in Fig. 3, two of each evolved strains with the highest cell growth rate, namely

225

PSA01009, PSA01020, PSA02004 and PSA02007 were selected for future evaluation in

226

comparison to fermentation using the initial strain PGC01003. These four evolved strains

227

were transferred for 10 generations in YPD medium containing 150 g/L initial glucose

228

concentration, then fermentations were carried out in medium using various initial glucose

229

concentrations range of 25-300 g/L. Results demonstrated that all of these four evolved

230

strains showed relatively high glucose tolerance i.e. cell growth was observed up to 200 g/L

231

initial glucose concentration. In addition, similar cell growth rate was observed as compared

232

to the growth in medium containing 25 g/L initial glucose concentration, suggesting the

233

evolved strains could resist high initial glucose concentration in fermentation medium. The

234

µmax profile showed a plateau of 0.23-0.30 h-1 from 25-200 g/L glucose, and dropped rapidly

235

to 0.06 ± 0.005 h-1 when the glucose concentration increased to 300 g/L. However, the

236

PGC01003 strain grew slowly with a maximum specific growth rate (µmax) reached only 0.09

237

h-1 in medium consisting of 25 g/L initial glucose concentration. Decrease in µmax was

238

observed in cultivation medium with increasing initial glucose concentration from 25 to

239

150 g/L, and eventually dropped to zero in medium consisting of 175 g/L initial glucose

240

concentration. Results from this study clearly demonstrate that the adaptive evolution by both

241

fermentation modes were efficient for enhancing cell growth rate and glucose uptake rate of

242

Y. lipolytica. Additionally, 5.5 ± 0.2 g/L SA was produced after 72 hours cultivation by these

243

evolved strains in medium containing 150 g/L initial glucose concentration. Therefore, the


Page 12 of 30

Page 13 of 30


244

PSA02004 strain was selected in the subsequent investigation using food waste hydrolysate

245

based on the highest experimentally observed cell dry weight. In order to assess the stability

246

of the evolved strain in glucose-rich YPD medium, the evolved strain was cultivated in YPG

247

medium containing glycerol as the major carbon source, and 1 mL of broth was then

248

transferred into YPD medium containing 150 g/L initial glucose concentration. After 24

249

hours cultivation, the resultant CDW was 4.67 ± 0.04 g/L, which indicates good stability of

250

the PSA02004 strain.

251 252

SA production from food waste hydrolysate by the evolved strain PSA02004

253

As shown in Figure 4, SA production from glucose-rich media by the evolved strain

254

PSA02004 was conducted in a 2-L fermenter using semi-defined YPD medium. Under

255

oxygen-limited condition i.e. aeration rate at 2 L/min aeration and stirring speed at 600 rpm,20

256

130.7 g/L glucose was consumed for production of 65.8 g/L SA with 22.5 g/L CDW after 96

257

hours (Fig. 4A). SA production significantly increased in batch fermentation in bench-top

258

fermenter. Apart from the high SA titer obtained in batch fermentation, high SA productivity

259

of 0.69 g/L/h was resulted using the PSA02004 strain. Moreover, the SA yield reached 0.50

260

g/g glucose which is equivalent to 0.76 mol/mol glucose, representing 76% of the theoretical

261

yield.10

262 263

Furthermore, its potential in SA production from glucose-rich food waste hydrolysate was

264

investigated, and hydrolysate from mixed food waste (i.e. leftover in restaurant) was used as

265

substrate. Food waste are potential sources of starch and protein-rich compounds that would

266

be used as nutrients in biotechnological processes, in which could be further hydrolysed by

267

the consortium of commercial enzymes consisting of glucoamylase, amylase and protease.2, 38

268

Food waste used in this study was collected from the same restaurant as our previous study,



269

which is rich in carbon and nitrogen sources of around 50% carbohydrate, 35–36% starch,

270

12–14% protein.32 After 24 hours hydrolysis by the commercial enzymes, 157.6 g/L glucose

271

and 1.3 g/L FAN were obtained in the food waste hydrolysate. Then the hydrolysate was used

272

as substrate with supplement of 5 g/L yeast extract in SA fermentation. After 126 hours

273

cultivation, 157 g/L glucose was consumed completely to produce 87.8 g/L SA with 16.1 g/L

274

of CDW (Fig. 4B). The SA yield and productivity reached 0.56 g/g glucose (equivalent to 85%

275

of theoretical yield) and 0.70 g/L/h, respectively. Compared to the batch fermentation using

276

defined medium, the SA titer, yield and productivity increased by 32%, 12% and 4%,

277

respectively. The highest SA yield in Y. lipolytica achieved was up to 85% of theoretical

278

yield. These results suggest that the evolved Y. lipolytica PSA02004 strain could achieve the

279

highest theoretical yield in SA production using food waste hydrolysate as feedstock.14, 21

280

Moreover, AA was completely depleted at the end of fermentation, which should be

281

consumed as a carbon source.20 These results also demonstrated that the use of evolved

282

Yarrowia yeast strain would open the opportunity for efficient and environmentally friendly

283

processes in commercial production of SA.

284 285

Since the first reported study for utilization of metabolic engineered Y. lipolytica for SA

286

production in 2010, the mechanism of partial or total loss of glucose metabolism ability has

287

not yet been revealed.10 Adaptive evolution has been demonstrated for enhancing glucose

288

uptake successfully by FBB. However, adaptive evolution strategy by FBB has several

289

disadvantages including highly-skilled labour, large volume of feeding medium and long

290

duration of fermentation process. In this study, the possibility of developing a simple and

291

efficient immobilised cell fermentation by shake flasks was investigated using adaptive

292

evolution strategy. After 21 days evolution, the glucose uptake rate, cell growth rate and

293

SA productivity of the SDH inactive Y. lipolytica were significantly increased, suggesting


Page 14 of 30

Page 15 of 30


294

that the glucose metabolism was restored successfully (Fig. 2). Furthermore, the experimental

295

results reported in this study show that the evolved strains have stable phenotype in

296

YPD medium containing high initial glucose concentration (i.e. up to 150 g/L) (Table 2, Fig.

297

3). To our knowledge, this is the first report that using adaptive evolution for SA production

298

from glucose-rich medium by engineered Y. lipolytica. Furthermore, the evolved strain

299

produced 87.9 g/L SA from food waste hydrolysate with 85% of theoretical yield, which is

300

the highest SA yield in yeast, to date (Fig. 4). This suggests that this evolved strain has a high

301

potential for SA fermentation using glucose-rich agricultural residues and food waste derived

302

feedstocks. Since there are around 3.7 billion tons of agricultural residues worldwide, with a

303

composition of about 40% cellulose39, 40. The annual global production of food wastes is 1.3

304

billion tons, and these food wastes contain around 30–60% of starch41, 42. These agricultural-

305

and food residues could potentially be hydrolysed as glucose-rich nutrient source that

306

representing a promising feedstock in biotechnological processes.2, 40, 43 Very recently, study

307

of this evolved yeast strain has been carried out in a novel mixed fruit and vegetable waste

308

biorefinery concept in SA fermentation by our group (Li et al. 2017).44 Moreover, in order to

309

decrease the cost of downstream processing, the evolutionary approach in this study would be

310

used for enhanced SA production in low pH environment using Y. lipolytica. As showcased

311

in our previous study, the PGC01003 strain has been reported for its ability in fermentative

312

SA production under low pH environment (i.e. pH 4).20 Concluding, the experimental results

313

reported in this study clearly demonstrate a proof of concept which paves the way for future

314

development of more process-friendly Y. lipolytica yeast strains in fermentative SA

315

production. This interesting approach would open the opportunity for generating useful

316

knowledge for the whole scientific yeast metabolic engineering community.

317



318

ABBREVIATIONS AND NOMENCLATURE

319

Acetic acid (AA), adenosine triphosphate (ATP), cell dry weight (CDW), dihydroxyacetone

320

phosphate (DHAP), flavin adenine dinucleotide (FAD), free amino nitrogen (FAN), fibrous-

321

bed bioreactor (FBB), generally recognized as safe (GRAS), nicotinamide adenine

322

dinucleotide (NAD), succinic acid (SA), succinate dehydrogenase (SDH), tricarboxylic acid

323

(TCA).

324 325

ACKNOWLEDGEMENT

326

The work described in this paper was fully supported by a grant from the City University of

327

Hong Kong [Project No. CityU 7004694].


Page 16 of 30

Page 17 of 30


328

REFERENCES

329

(1)

Koutinas, A. A.; Vlysidis, A.; Pleissner, D.; Kopsahelis, N.; Garcia, I. L.; Kookos, I. K.;

330

Papanikolaou, S.; Kwan, T. H.; Lin, C. S. K., Valorization of industrial waste and by-

331

product streams via fermentation for the production of chemicals and biopolymers.

332

Chem. Soc. Rev. 2014, 43, 2587-2627.

333

(2)

Lin, C. S. K.; Pfaltzgraff, L. A.; Herrero-Davila, L.; Mubofu, E. B.; Abderrahim, S.;

334

Clark, J. H.; Koutinas, A. A.; Kopsahelis, N.; Stamatelatou, K.; Dickson, F.;

335

Thankappan, S.; Mohamed, Z.; Brocklesby, R.; Luque, R., Food waste as a valuable

336

resource for the production of chemicals, materials and fuels. Current situation and

337

global perspective. Energy Environ. Sci. 2013, 6, 426-464.

338

(3)

Werpy, T.; Petersen, G. Top value added chemicals from biomass: Results of screening

339

for potential candidates from sugars and synthesis gas; Vol 1; US Department of

340

Energy: Oakridge, USA, 2004.

341

(4)

Bozell, J. J.; Petersen, G. R., Technology development for the production of biobased

342

products from biorefinery carbohydrates-the US Department of Energy's "Top 10"

343

revisited. Green Chem. 2010, 12, 539-554.

344

(5)

Taylor, R.; Nattrass, L.; Alberts, G.; Robson, P.; Chudziak, C.; Bauen, A.; Libelli, I.;

345

Lotti, G.; Prussi, M.; Nistri, R. From the Sugar Platform to biofuels and biochemicals:

346

Final report for the European Commission Directorate-General Energy; E4tech/Re-

347

CORD/Wageningen UR: 2015.

348

(6)

Natural versus metabolic engineered producers. Process Biochem. 2010, 45, 1103-1114.

349 350

Beauprez, J. J.; De Mey, M.; Soetaert, W. K., Microbial succinic acid production:

(7)

Cukalovic, A.; Stevens, C. V., Feasibility of production methods for succinic acid

351

derivatives: a marriage of renewable resources and chemical technology. Biofuels

352

Bioprod. Biorefin. 2008, 2, 505-529.



353

(8)

Zhu, L; Xia, S.; Wei, L.; Li, H.; Yuan, Z.; Tang, Y., Enhancing succinic acid

354

biosynthesis in Escherichia coli by engineering its global transcription factor, catabolite

355

repressor/activator (Cra). Sci. Rep. 2016, 6, 36526.

356

(9)

Yan, D.; Wang, C.; Zhou, J.; Liu, Y.; Yang, M.; Xing, J., Construction of reductive

357

pathway in Saccharomyces cerevisiae for effective succinic acid fermentation at low

358

pH value. Bioresour. Technol. 2014, 156, 232-239.

359

(10) Yuzbashev, T. V.; Yuzbasheva, E. Y.; Sobolevskaya, T. I.; Laptev, I. A.; Vybornaya, T.

360

V.; Larina, A. S.; Matsui, K.; Fukui, K.; Sineoky, S. P., Production of succinic acid at

361

low pH by a recombinant strain of the aerobic yeast Yarrowia lipolytica. Biotechnol.

362

Bioeng. 2010, 107, 673-682.

363

(11) Ito, Y.; Hirasawa, T.; Shimizu, H., Metabolic engineering of Saccharomyces cerevisiae

364

to improve succinic acid production based on metabolic profiling. Biosci. Biotechnol.

365

Biochem. 2014, 78, 151-159.

366

(12) Otero, J. M.; Cimini, D.; Patil, K. R.; Poulsen, S. G.; Olsson, L.; Nielsen, J., Industrial

367

systems biology of Saccharomyces cerevisiae enables novel succinic acid cell factory.

368

PloS ONE 2013, 8, e54144.

369

(13) Agren, R.; Otero, J. M.; Nielsen, J., Genome-scale modeling enables metabolic

370

engineering of Saccharomyces cerevisiae for succinic acid production. J. Ind.

371

Microbiol. Biotechnol. 2013, 40, 735-747.

372

(14) Ahn, J. H.; Jang, Y. S.; Lee, S. Y., Production of succinic acid by metabolically engineered microorganisms. Curr. Opin. Biotechnol. 2016, 42, 54-66.

373 374 375

(15)

Van De Graaf, M. J.; Valianpoer, F.; Fiey, G.; Delattre, L.; Schulten, E. A. M. Process for the crystallization of succinic acid. US20150057425A1, 2015.


Page 18 of 30

Page 19 of 30


376

(16) Jost, B.; Holz, M.; Aurich, A.; Barth, G.; Bley, T.; Mueller, R. A., The influence of

377

oxygen limitation for the production of succinic acid with recombinant strains of

378

Yarrowia lipolytica. Appl. Microbiol. Biotechnol. 2015, 99, 1675-1686.

379

(17) Kamzolova, S. V.; Vinokurova, N. G.; Shemshura, O. N.; Bekmakhanova, N. E.;

380

Lunina, J. N.; Samoilenko, V. A.; Morgunov, I. G., The production of succinic acid by

381

yeast Yarrowia lipolytica through a two-step process. Appl. Microbiol. Biotechnol.

382

2014, 98, 7959-7969.

383

(18) Kamzolova, S. V.; Vinokurova, N. G.; Dedyukhina, E. G.; Samoilenko, V. A.; Lunina,

384

J. N.; Mironov, A. A.; Allayarov, R. K.; Morgunov, I. G., The peculiarities of succinic

385

acid production from rapeseed oil by Yarrowia lipolytica yeast. Appl. Microbiol.

386

Biotechnol. 2014, 98, 4149-4157.

387

(19) Kamzolova, S. V.; Yusupova, A. I.; Vinokurova, N. G.; Fedotcheva, N. I.;

388

Kondrashova, M. N.; Finogenova, T. V.; Morgunov, I. G., Chemically assisted

389

microbial production of succinic acid by the yeast Yarrowia lipolytica grown on

390

ethanol. Appl. Microbiol. Biotechnol. 2009, 83, 1027-1034.

391

(20) Gao, C.; Yang, X.; Wang, H.; Rivero, C. P.; Li, C.; Cui, Z.; Qi, Q.; Lin, C. S. K.,

392

Robust succinic acid production from crude glycerol by using engineered Yarrowia

393

lipolytica. Biotechnol. Biofuels 2016, 9, 179.

394

(21) Li, C.; Yang, X.; Gao, S.; Wang, H.; Lin, C. S. K., High efficiency succinic acid

395

production from glycerol via in situ fibrous bed bioreactor with an engineered Yarrowia

396

lipolytica. Bioresour. Technol. 2017, 225, 9-16.

397

(22) Zhu, X.; Tan, Z.; Xu, H.; Chen, J.; Tang, J.; Zhang, X., Metabolic evolution of two

398

reducing equivalent-conserving pathways for high-yield succinate production in

399

Escherichia coli. Metab. Eng. 2014, 24, 87-96.



400

(23) Singh, A.; Soh, K. C.; Hatzimanikatis, V.; Gill, R. T., Manipulating redox and ATP

401

balancing for improved production of succinate in E. coli. Metab. Eng. 2011, 13, 76-81.

402

(24) Singh, A.; Lynch, M. D.; Gill, R. T., Genes restoring redox balance in fermentation-

403

deficient E. coli NZN111. Metab. Eng. 2009, 11, 347-354.

404

(25) Zhang, X. L.; Jantama, K.; Moore, J. C.; Jarboe, L. R.; Shanmugam, K. T.; Ingram, L.

405

O., Metabolic evolution of energy-conserving pathways for succinate production in

406

Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 20180-20185.

407 408

(26) Wu, Z. T.; Yang, S. T., Extractive fermentation for butyric acid production from glucose by Clostridium tyrobutyricum. Biotechnol. Bioeng. 2003, 82, 93-102.

409

(27) Jiang, L.; Li, S.; Hu, Y.; Xu, Q.; Huang, H., Adaptive evolution for fast growth on

410

glucose and the effects on the regulation of glucose transport system in Clostridium

411

tyrobutyricum. Biotechnol. Bioeng. 2012, 109, 708-718.

412

(28) Li, H. G.; Ma, X. X.; Zhang, Q. H.; Luo, W.; Wu, Y. Q.; Li, X. H., Enhanced butanol

413

production by solvent tolerance Clostridium acetobutylicum SE25 from cassava flour in

414

a fibrous bed bioreactor. Bioresour. Technol. 2016, 221, 412-418.

415

(29) Sanderson, K., Lignocellulose: a chewy problem. Nature 2011, 474, S12-S14.

416

(30) Kawaguchi, H.; Hasunuma, T.; Ogino, C.; Kondo, A., Bioprocessing of bio-based

417

chemicals produced from lignocellulosic feedstocks. Curr. Opin. Biotechnol. 2016, 42,

418

30-39.

419

(31) Sun, Z.; Li, M.; Qi, Q.; Gao, C.; Lin, C. S. K., Mixed food waste as renewable

420

feedstock in succinic acid fermentation. Appl. Biochem. Biotechnol. 2014, 174, 1822-

421

1833.

422

(32) Kwan, T. H.; Hu, Y. Z.; Lin, C. S. K., Valorisation of food waste via fungal hydrolysis

423

and lactic acid fermentation with Lactobacillus casei Shirota. Bioresour. Technol. 2016,

424

217, 129-136.


Page 20 of 30

Page 21 of 30

425 426


(33) Norman Leslie Kent, A. D. E., Technology of cereals: an introduction for students of food science and agriculture. 4th ed.; Oxford: Pergamon, 1994.

427

(34) Hao, H. X.; Khalimonchuk, O.; Schraders, M.; Dephoure, N.; Bayley, J. P.; Kunst, H.;

428

Devilee, P.; Cremers, C.; Schiffman, J. D.; Bentz, B. G.; Gygi, S. P.; Winge, D. R.;

429

Kremer, H.; Rutter, J., SDH5, a gene required for flavination of succinate

430

dehydrogenase, is mutated in paraganglioma. Science 2009, 325, 1139-1142.

431

(35) Oyedotun, K. S.; Lemire, B. D., The quaternary structure of the Saccharomyces

432

cerevisiae succinate dehydrogenase - Homology modeling, cofactor docking, and

433

molecular dynamics simulation studies. J. Biol. Chem. 2004, 279, 9424-9431.

434

(36) Workman, M.; Holt, P.; Thykaer, J., Comparing cellular performance of Yarrowia

435

lipolytica during growth on glucose and glycerol in submerged cultivations. AMB

436

Express 2013, 3, 58

437 438

(37) Ryu, S.; Hipp, J.; Trinh, C. T., Activating and elucidating metabolism of complex sugars in Yarrowia lipolytica. Appl. Environ. Microbiol. 2015, 82, 1334-1345.

439

(38) Pleissner, D.; Kwan, T. H.; Lin, C. S. K., Fungal hydrolysis in submerged fermentation

440

for food waste treatment and fermentation feedstock preparation. Bioresour. Technol.

441

2014, 158, 48-54.

442 443

(39) Bentsen, N. S.; Felby, C.; Thorsen, B. J., Agricultural residue production and potentials for energy and materials services. Prog. Energy Combust. Sci. 2014, 40, 59-73.

444

(40) Pleissner, D.; Venus, J., Agricultural Residues as Feedstocks for Lactic Acid

445

Fermentation. In Green Technologies for the Environment, American Chemical Society:

446

2014; Vol. 1186, pp 247-263.

447

(41) Gustavsson, J.; Cederberg, C.; Sonesson, U.; van Otterdijk, R.; Meybeck, A. Global

448

food losses and food waste. Extent, causes and prevention; Food and Agriculture

449

Organization of the United Nations: Rome, 2011.



450 451

(42) Li, S.; Yang, X., Biofuel production from food wastes. In Handbook of Biofuels Production (2nd Edition), Woodhead Publishing: 2016; pp 617-653.

452

(43) Pleissner, D.; Qi, Q.; Gao, C.; Rivero, C. P.; Webb, C.; Lin, C. S. K.; Venus, J.,

453

Valorization of organic residues for the production of added value chemicals: A

454

contribution to the bio-based economy. Biochem. Eng. J. 2016, 116, 3-16.

455

(44) Li, C.; Yang, X.; Gao, S.; Lin, C. S. K., Production and optimization of fruit and

456

vegetable agro-waste for efficient succinic acid with an engineered Yarrowia lipolytica.

457

J. Cleaner Prod. 2017. (under review)


Page 22 of 30

Page 23 of 30


458

Figures captions

459

Fig. 1 Metabolic pathways showing the flow of NAD+/NADH and FAD/FADH2 with

460

utilisation of glucose and glycerol in Y. lipolytica PGC01003. The red cross represents the

461

oxidation of SA into fumaric acid, which was blocked by deletion of Ylsdh5 in Y. lipolytica

462

PGC01003. The metabolic pathway from glucose to GAP in glycolysis was indicated by

463

three arrows, in which neither NAD+/NADH nor FAD/FADH2 pathways were accompanied.

464

DHAP: dihydroxyacetone phosphate, GAP: glyceraldehyde-3-phosphate, Glycerol-3-P:

465

Glycerol 3-phosphate, Q: ubiquinone.

466 467

Fig. 2 Comparison of adaptive evolution progress of Y. lipolytica in free cell fermentation and

468

immobilised cell fermentation. Relative growth rate (A), in which the cell growth rate of the

469

first generation was set as 100%. [Relative growth rate = (cell growth rate in any

470

generation)/(cell growth rate in the first generation) ×100%]. Glucose consumption rate (B)

471

and SA productivity (C) were presented as the average value in each generation.

472 473

Fig. 3 Comparison of maximum specific growth rate of the adapted and wild-type strains in

474

defined media with various initial glucose concentrations of 25-300 g/L in shake flask

475

fermentation.

476 477

Fig. 4 Bench-top scale fermentation profile using the adapted strain PSA02004 in YPD

478

medium

(A)

and

food

waste


hydrolysate

(B).


Page 24 of 30

Table 1. Fermentative SA production by Y. lipolytica PGC01003 in YPG and YPD media. Fermentation CDW Medium time (h) (g/L)

Glycerol or glucose consumption (g/L)

SA titer (g/L)

SA yield (g/g)

SA productivity (g/L/h)

AA titer (g/L)

YPG

48

4.0

18.6

5.4

0.29

0.06

3.8

YPD

84

1.8

5.7

1.9

0.33

0.02

4.4


Page 25 of 30


Table 2. Summary of CDW and µmax of the evolved strains in YPD medium with 150 g/L initial glucose concentration. Strain

CDW(g/L)

µmax (h-1)

PSA01002

2.91

0.55

PSA01005

2.88

0.47

PSA01009

2.93

0.67

PSA01010

2.89

0.46

PSA01011

2.92

0.47

PSA01020

2.89

0.74

PSA02004

2.95

0.72

PSA02005

2.78

0.61

PSA02006

2.85

0.55

PSA02007

2.81

0.62

PSA02008

2.81

0.61

PSA02011

2.81

0.61



Page 26 of 30

Fig.1 Glucose

Glycerol NADH

GAP

NAD+

Glycerol-3-P

DHAP

NAD+ NADH

Pyruvate

FADH2

NAD+

FAD

NADH

Acetyl-CoA QH2 Oxaloacetate

NADH

Q

Citrate

NAD+

Isocitrate

Malate

NAD+

TCA cycle NADH

Fumarate FADH2 FAD

ketoglutarate NAD+

Succinate

Succinyl-CoA

NADH


Mitochondrial

Page 27 of 30


Fig. 2 ˚

175

300

150

250

125

200

100 150 75 100

50

50

25 0

Relative growth rate (%)

Glucose concentration (g/L)

A

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Generation number

0.4


175 150

0.3

125 100

0.2 75 50

0.1

25 0

0.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Glucose consumption rate (g/L/h)

B

Generation number

C 0.20 Glucose concentration 150 Free cell mode 125

0.15

Immobilised cell mode

100 0.10 75 50

0.05

25 0

0.00 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Generation number


SA productivity (g/L/h)


175


Fig. 3


Page 28 of 30

Page 29 of 30


Fig. 4

20

90

15

60

10

30

5 0

24

48 72 Time (h)

96

0

30 Glucose SA AA CDW

150 Glucose, SA, AA (g/L)

25

120

0

180

120

25 20

90

15

60

10

30

5

0

0

24


48

72

Time (h)

96

120

0

CDW (g/L)

30

Glucose SA AA CDW

150 Glucose, SA, AA (g/L)

B

˚

180

CDW (g/L)

A


TOC Graphic

Page 30 of 30

Evolved strains Adaptive evolution Y. lipolytica PGC01003

SA production Food waste

Hydrolysis

ACS Paragon Plus Environment

Food waste hydrolysate

Restoring of Glucose Metabolism of Engineered Yarrowia

Recommend Documents